Materials and methods to inhibit hodgkin and reed sternberg cell growth

ABSTRACT

Materials and methods are disclosed for modulating proliferation of cell types associated with Hodgkin&#39;s disease through inhibition of IL-13 and components in IL-13 associated signal transduction pathways. Methods to identify inhibitors, compositions comprising the inhibitors, and methods using the inhibitors to treat Hodgkin&#39;s disease are also disclosed.

FIELD OF THE INVENTION

The present invention relates generally to materials and methods forinhibition of Hodgkin and Reed Sternberg cell proliferation.

BACKGROUND OF THE INVENTION

Hodgkin's disease (HD) is unique among human lymphomas in that the tumorcells, known as Hodgkin and Reed Sternberg (H/RS) cells, are exceedinglyrare, generally representing 0.1-1% of the total cell population withinlymphoma tissue [Drexler, Leukemia and Lymphoma. 8:283-313 (1992);Drexler, Leukemia and Lymphoma. 9:1-25 (1993); Kadin, Current Opinion inOncology 6:456:463 (1994); Cossman, et al., Lab. Invest. 78:229-235(1998)]. As a result, investigation of H/RS cells has been impeded bytheir low frequency. PCR-based assays of DNA from single H/RS cells haverevealed rearranged immunoglobulin genes and somatic mutations,suggesting that H/RS cells are clonal and may be derived from germinalcenter B cells [Kanzler, et al., J Exp Med 184:1495-505 (1996); Kÿppers,et al., Annu Rev Immunol 16:471-93 (1998)]. The precise pathogenesis ofHD, however, remains to be determined.

Previous observations suggest that the proliferation and survival ofHD-derived cells depends on cytokine signaling. It is well establishedthat the unique histology and eosinophilia of HD tissues, and secondarysymptoms in the patient such as fever, weight loss, and night sweats,are induced by a pathological pattern of cytokine secretion [Drexler,Leukemia and Lymphoma. 8:283-313 (1992); Drexler, Leukemia and Lymphoma.9:1-25 (1993)]. For example, overexpression of IL-5 in H/RS cells hasbeen previously demonstrated by in situ hybridization, but only in HDpatients exhibiting eosinophilia [Samoszuk, et al., Blood 75:13-16(1990); Samoszuk, Blood 79:1518-22 (1992)]. To date, no cytokine hasbeen consistently reported as being overexpressed in HD-derived celllines or in primary H/RS cells.

Evidence for a role for IL-13 in the etiology of HD is indirect. IgE iselevated in HD tissues and serum samples from HD patients [Samoszuk,Blood 79:1518-22 (1992); Thomas, et al., Ann Allergy 37:416-19 (1976)],and IL-13 is known to promote Ig class switching to IgE. IL-13-deficientmice exhibit lower basal levels of serum IgE [McKenzie, et al., Immunity9:423-432 (1998)]. Furthermore, studies of IL-4 deficient, IL-13transgenic mice have demonstrated that IL-13 can promote class switchingto IgE independently of IL-4 [Emson, et al., J. Exp. Med. 188:399-404(1998)], emphasizing that IL-4 and IL-13 have distinct roles inregulating B cell functions.

IL-13 is a T cell-derived cytokine with immunomodulatory andanti-inflammatory properties [Minty, et al., Nature 362:248-250 (1993)].The biological effects of IL-13 on B cells, macrophages, and monocytesare very similar to those of IL-4, probably because the IL-4 and IL-13receptors share a common a chain. In B cells, IL-13 promotesproliferation, differentiation, and Ig heavy chain class switching toIgE and IgG4 [Zurawski, et al., Immunology Today 15:19-26 (1994)].Proliferation results from a signaling pathway in which the engagementof the IL-13 receptor activates JAK1, which in turn activates STAT6[Lin, et al., Immunity 2:331-339 (1995)].

Other aspects of the HD phenotype may also be attributable to theeffects of IL-13. A recent study of IL-13-deficient mice has shown thatcultures of type 2 helper T (Th2) cells from these animals producesignificantly reduced levels of IL-4, IL-5, and IL-10 compared to thewild type, suggesting an important role for IL-13 as a regulator of Th2cell commitment [McKenzie, et al., Immunity 9:423-432 (1998)]. If IL-13is also important for promoting the differentiation of Th2 cells inhumans, it could explain why H/RS cells (which secrete IL-13) aresurrounded by Th2 cells in HD biopsies. In addition, because fibroblastsexpress the IL-13 receptor and can be activated by IL-13 [Doucet, etal., J Clin Invest 101:2129-2139 (1998)], the secretion of IL-13 by H/RScells may underlie the pathogenesis of the fibrosis observed in nodularsclerosis HD.

Thus, there exists a need in the art to identify specific growth factorsthat participate in the pathogenesis of HD. In particular,identification of cytokines that stimulate H/RS cell proliferation willfacilitate development of methods for therapeutic invention, by way of(i) modulating expression of the cytokine(s), (ii) modulating biologicalactivity of the cytokine(s), and (iii) modulating both expression andbiological activity.

SUMMARY OF THE INVENTION

The present invention provides materials and methods for inhibitingproliferation of cell types associated with Hodgkin's disease. Inparticular, the invention contemplates inhibiting proliferation of ReedSternberg cells found in individuals suffering from Hodgkin disease.Reed Sternberg cells associated with Hodgkin's disease are referred toherein as H/RS cells.

The invention comprehends various methods by which proliferation of H/RScells can be modulated, preferably through mechanisms that modulateexpression, secretion, stimulation, activation, and/or biologicalactivity of IL-13 and/or its cognate IL-13 receptor, as well ascomponents in signal transduction pathways associated with IL-13 and theIL-13 receptor. In one aspect, the invention provides methods forinhibiting IL-13 dependent proliferation of Hodgkin and Reed Sternberg(H/RS) cell types comprising the step of contacting the H/RS cells witha compound that inhibits biological activity of IL-13. In a preferredembodiment, methods are provided wherein the compound that inhibitsIL-13 biological activity is an antibody. It is preferred that theantibody is a monoclonal antibody that specifically recognizes and bindsto IL-13, and most preferably, the monoclonal antibody is a humanantibody, however, humanized antibodies and non-human antibodies arealso comprehended by the invention. The term “specifically recognizes orbinds” as used herein indicates antibodies of the invention bind to onlyIL-3, IL-13 receptor, or naturally occurring variants thereof, despiteany sequence similarity, homology, or identity conserved in otherpolypeptides.

In another aspect, the invention provides methods for inhibiting IL-13dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell typescomprising the step of contacting the H/RS cell types with a compoundthat inhibits biological activity of IL-13 receptors expressed on theH/RS cell types. In a preferred embodiment, methods are provided whereinthe compound that inhibits IL-13 receptor biological activity is anantibody. It is preferred that the antibody is a monoclonal antibodythat specifically recognizes and binds to the IL-13 receptor, and mostpreferably, the monoclonal antibody is a human antibody, however,humanized antibodies and non-human antibodies are also comprehended bythe invention.

Alternatively, the invention provides methods for inhibiting IL-13dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell typescomprising the step of contacting the H/RS cells with a compound thatinhibits expression of IL-13. In a preferred embodiment, methods of theinvention are provided wherein the compound that inhibits IL-13expression is an antisense polynucleotide that specifically recognizes apolynucleotide encoding IL-13. In another preferred embodiment, methodsare provided wherein the compound that inhibits IL-13 expression is aribozyme that binds to and acts specifically on polynucleotides encodingIL-13. The terms “act specifically on” and “specifically recognizes orbinds” as used herein indicates that anti-sense and ribozymes of theinvention bind to and act only on polynucleotides that encode IL-13, theIL-13 receptor, or naturally occurring variants thereof, despite anysequence similarity, homology, or identity conserved in polynucleotidesencoding other polypeptides.

Similarly, the invention also provides methods for inhibiting IL-13dependent proliferation of Hodgkin and Reed Sternberg (H/RS) cell typescomprising the step of contacting the H/RS cells with a compound thatinhibits expression of IL-13 receptor. In a preferred embodiment,methods of the invention are provided wherein the compound that inhibitsIL-13 receptor expression is an antisense polynucleotide thatspecifically recognizes a polynucleotide encoding the IL-13 receptor. Inanother preferred embodiment, methods are provided wherein the compoundthat inhibits IL-13 receptor expression is a ribozyme that binds to andacts specifically on polynucleotides encoding the IL-13 receptor.

The invention comprehends methods for identifying a compound thatinhibits IL-13 dependent proliferation of Hodgkin and Reed Sternberg(H/RS) cell types comprising the steps of: a) measuring binding betweenIL-13 with IL-13 receptor in the presence and absence of a candidateinhibitor compound; and b) identifying the candidate compound as aninhibitor when binding between IL-13 and the IL-13 receptor is less inthe presence of the candidate compound than in the absence of thecandidate compound. The invention also provides methods for identifyinga compound, or combination of compounds, that inhibits IL-13 dependentproliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprisingthe steps of: a) contacting IL-13 with IL-13 receptor; b) measuringbinding between IL-13 and the IL-13 receptor; c) repeating step (a) inthe presence of a candidate inhibitor compound or compounds; d)repeating step (b) in the presence of the candidate compound orcompounds; and e) identifying the candidate compound or compounds as aninhibitor when binding between IL-13 and the IL-13 receptor is less inthe presence of the candidate compound than in the absence of thecandidate compound. In a preferred embodiment of the method, IL-13receptor is expressed on the cell surface of the H/RS cell types.

The invention also provide methods to identify a compound that inhibitsIL-13 dependent proliferation of Hodgkin and Reed Sternberg (H/RS) celltypes comprising the steps of: a) measuring IL-13 expression fromgrowing H/RS cells in the presence and absence of a candidate inhibitorcompound; and b) identifying the candidate compound as an inhibitor whenlower levels of IL-13 expression are measured in the presence of thecandidate compound than in an absence of the candidate compound. In oneaspect, the invention provides methods to identify a compound orcombination of compounds that inhibits IL-13 dependent proliferation ofHodgkin and Reed Sternberg (H/RS) cell types comprising the steps of: a)growing H/RS cell types; b) measuring IL-13 expression; c) incubatingthe cell with a candidate inhibitor compound or compounds; d) measuringIL-13 expression with the candidate compound or compounds; and e)identifying the candidate compound or compounds as an inhibitor whenlower levels of IL-13 expression are measured with the candidatecompound or compounds than in an absence of the candidate compound orcompounds. In a preferred embodiment of the method, the candidateinhibitor compound is an anti-sense polynucleotide that specificallybinds to a polynucleotide encoding IL-13.

In methods of the invention in which IL-13 or IL13 receptor biologicalactivity is inhibited, numerous modes of action for a candidateinhibitor are contemplated. In one aspect, inhibitors are provided thatbind and neutralize IL-13. Preferably, an inhibitor acting by thismechanism prevents interaction between IL-13 and the IL-13 receptor. Theinvention comprehends mechanisms by which the inhibitor bindinginteraction induces a conformational change in IL-13, and binding thatresults in stearic hindrance preventing receptor recognition. Theinvention further comprehends inhibitors of the IL-13 receptorbiological activity, which like IL-13 inhibitors, can function in avariety of ways. In one aspect, the IL-13 receptor inhibitor binds andneutralizes IL-13 activity. Preferably, the inhibitor would preventinteraction of the receptor with IL-13, or may prevent interaction ofthe IL-13 receptor with some downstream component of an IL-13 metabolicpathway that is required for, or participates in, H/RS cellproliferation. For example, it is known that IL-13 receptor activatesJAK1 and the invention therefore embraces inhibitors that prevent thisactivation, directly or indirectly.

The invention further embraces methods to inhibit activity of, orinteraction between, components in IL-13 pathways downstream from theIL-13 receptor that are required for, or at least participate in, H/RScell proliferation.

In identification of inhibitors, the invention provides various sourcesfor candidate compounds. There are a number of different libraries usedfor the identification of small molecule modulators, including, (1)chemical libraries, (2) natural product libraries, and (3) combinatoriallibraries comprised of random peptides, oligonucleotides or organicmolecules. Chemical libraries consist of structural analogs of knowncompounds or compounds that are identified as “hits” or “leads” vianatural product screening. Natural product libraries are collections ofmicroorganisms, animals, plants, or marine organisms which are used tocreate mixtures for screening by: (1) fermentation and extraction ofbroths from soil, plant, or marine microorganisms or (2) extraction ofplants or marine organisms. Natural product libraries includepolyketides, non-ribosomal peptides, and variants (non-naturallyoccurring) thereof. For a review, see Science 282:63-68 (1998).Combinatorial libraries are composed of large numbers of peptides,oligonucleotides, or organic compounds as a mixture. They are relativelyeasy to prepare by traditional automated synthesis methods, PCR, cloningor proprietary synthetic methods. Of particular interest are peptide andoligonucleotide combinatorial libraries. Still other libraries ofinterest include peptide, protein, peptidomimetic, multiparallelsynthetic collection, recombinatorial, and polypeptide libraries. For areview of combinatorial chemistry and libraries created therefrom, seeMyers, Curr. Opion. Biotechnol. 8:701-707 (1997). Identification ofmodulators (“hits”) through use of the various libraries describedherein permits modification of the candidate “hit” (or “lead”) tooptimize the capacity of the “hit” to modulate activity. Methods of theinvention are particularly amenable to high throughput screening (HTS)assays wherein large numbers of candidate inhibitors can been screensimultaneously, preferably by automated mechanisms.

In another aspect, the invention provides antibodies that inhibit IL-13or IL-13 receptor activity. The invention provides monoclonal andpolyclonal antibodies, single chain antibodies, chimeric antibodies, andCDR-grafted antibodies, including compounds which include CDR sequenceswhich specifically recognize a polypeptide of the invention. In apreferred embodiment, the invention provides human antibodies thatspecifically recognize IL-13, IL-13 receptor, other components in IL-13pathways that participate in H/RS cell proliferation, and naturallyoccurring variants thereof. Human antibodies are produced and identifiedaccording to methods described in WO93/11236, published Jun. 20, 1993,which is incorporated herein by reference in its entirety. The term“specifically recognize” indicates that the variable regions of theantibodies recognize and bind, for example IL-13 or IL-13 receptorpolypeptides, exclusively (i.e., able to distinguish IL-13 the IL-13receptor, or naturally occurring variants thereof, from closely relatedpolypeptides despite sequence identity, homology, or similarity found inthe structure of the polypeptides), but may also interact with otherproteins (for example, S. aureus protein A or other antibodies in ELISAtechniques) through interactions with sequences outside the variableregion of the antibodies, and in particular, in the constant region ofthe molecule. Screening assays to determine binding specificity of anantibody of the invention are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies that recognizeand bind fragments of, for example, IL-13 or IL-13 receptor polypeptidesof the invention, are also contemplated, provided that the antibodiesare first and foremost specific for intact (i.e., “unfragmented”)polypeptides. As with antibodies that are specific for full length IL-13or IL-13 receptor polypeptides (or other polypeptides of the invention),antibodies that recognize IL-13 or IL-13 receptor fragments are thosewhich can distinguish 11-13 or IL-13 receptor polypeptides from otherpolypeptides despite inherent sequence and structural identity,homology, or similarity.

As still another aspect, the invention provides antisensepolynucleotides which inhibit translation of a polynucleotide encodingIL-13, IL-13 receptor, other components of an IL-13 pathway thatparticipate in H/RS cell proliferation, and naturally occurring variantsthereof. Full length and fragment anti-sense polynucleotides areprovided. The worker of ordinary skill will appreciate that fragmentanti-sense molecules of the invention include (i) those whichspecifically recognize and hybridize to, for example either IL-13 orIL-13 receptor, RNA (as determined by sequence comparison of DNAencoding IL-13 or the IL-13 receptor to DNA encoding other knownmolecules and identification of characteristic or signature sequences)as well as (ii) those which recognize and hybridize to RNA encodingvariants of the, for example, IL-13 or IL-13 receptor family ofproteins. Antisense polynucleotides that hybridize to RNA encodingrelated members of the IL-13 or IL-13 receptor families of proteins arealso identifiable through sequence comparison to identifycharacteristic, or signature, sequences for either family of molecules.

The invention further contemplates methods to modulate IL-13,IL-13receptor, other components of IL-13 pathway that participate in H/RScell proliferation, and naturally occurring variants thereof, expressionthrough use of ribozymes. For a review, see Gibson and Shillitoe, Mol.Biotech. 7:125-137 (1997). Ribozyme technology can be utilized toinhibit translation of, for example, IL-13 or IL-13 receptor mRNA in asequence specific manner through (i) the hybridization of acomplementary RNA to a target mRNA and (ii) cleavage of the hybridizedmRNA through nuclease activity inherent to the complementary strand.Ribozymes can be identified by empirical methods, but more preferablyare specifically designed based on accessible sites on the target mRNA(Bramlage, et al., Trends in Biotech 16:434-438 (1998). Delivery ofribozymes to target cells can be accomplished using either exogenous orendogenous delivery techniques well known and routinely practiced in theart. Exogenous delivery methods can include use of targeting liposomesor direct local injection. Endogenous methods include use of viralvectors and non-viral plasmids.

Ribozymes can specifically modulate expression of Il-13, IL-13 receptor,other components of an IL-13 pathway that participate in H/RS cellproliferation, and naturally occurring variants thereof, when designedto be complementary to regions unique to a polynucleotide encodingeither polypeptide. “Specifically modulate” therefore is intended tomean that ribozymes of the invention recognizes only a polynucleotideencoding, for example, either IL-13 or the IL-13 receptor. Similarly,ribozymes can be designed to modulate expression of all or some of theproteins closely related to either IL-13 or the IL-13 receptor.Ribozymes of this type are designed to recognize polynucleotidesequences conserved in all or some of the polynucleotides which encodethe family of proteins.

The invention further embraces methods to modulate transcription ofIL-13, IL-13 receptor, other components of an IL-13 pathway thatparticipate in H/RS cell proliferation, and naturally occurring variantsthereof, through use of oligonucleotide-directed triplet helixformation. For a review, see Lavrovsky, et al., Biochem. Mol. Med.62:11-22 (1997). Triplet helix formation is accomplished using sequencespecific oligonucleotides which hybridize to double stranded DNA in themajor groove as defined in the Watson-Crick model. Hybridization of asequence specific oligonucleotide can thereafter modulate activity ofDNA-binding proteins, including, for example, transcription factors andpolymerases. Preferred target sequences for hybridization includepromoter and enhancer regions to permit regulated transcription ofIL-13, IL-13 receptor, other components of an IL-13 pathway thatparticipate in H/RS cell proliferation, and naturally occurring variantsthereof. Oligonucleotides which are capable of triplet helix formationare also useful for site-specific covalent modification of target DNAsequences. Oligonucleotides useful for covalent modification are coupledto various DNA damaging agents as described in Lavrovsky, et al.[supra].

The invention further provides methods for treating Hodgkin's Diseasecomprising the steps of administering to an individual in need thereof atherapeutically effective amount of a compound that inhibits IL-13biological activity. Similarly, the invention contemplates methods fortreating Hodgkin's Disease comprising the steps of administering to anindividual in need thereof a therapeutically effective amount of acompound that inhibits IL-13 expression. In a preferred method, theinhibitor is an antisense polynucleotide that specifically binds apolynucleotide encoding IL-13 or an antibody that specificallyrecognizes an IL-13 polypeptide.

The invention also comprehends methods for treating Hodgkin's Diseasecomprising the step of administering to an individual in need thereof atherapeutically effective amount of a compound that inhibits IL-13receptor biological activity. In another aspect, the invention providesmethods for treating Hodgkin's Disease comprising the step ofadministering to an individual in need thereof a therapeuticallyeffective amount of a compound that inhibits IL-13 receptor expression.

Inhibitors of the invention can be administered in combination with apharmaceutically acceptable (i.e., sterile and non-toxic) liquid,semisolid, or solid diluents that serve as pharmaceutical vehicles,excipients, or media. Any diluent known in the art may be used.Exemplary diluents include, but are not limited to, polyoxyethylenesorbitan monolaurate, magnesium stearate, methyl- andpropylhydroxybenzoate, talc, alginates, starches, lactose, sucrose,dextrose, sorbitol, mannitol, gum acacia, calcium phosphate, mineraloil, cocoa butter, and oil of theobroma.

The inhibitor compositions can be packaged in forms convenient fordelivery. The compositions can be enclosed within a capsule, sachet,cachet, gelatin, paper, or other container. These delivery forms arepreferred when compatible with entry of the composition into therecipient organism and, particularly, when the composition is beingdelivered in unit dose form. The dosage units can be packaged, e.g., intablets, capsules, suppositories or cachets.

The inhibitor compositions may be introduced into the subject by anyconventional method including, e.g., by intravenous, intradermal,intramuscular, intramammary, intraperitoneal, or subcutaneous injection;by oral, sublingual, nasal, anal, vaginal, or transdermal delivery; orby surgical implantation, e.g., embedded under the splenic capsule or inthe cornea. The treatment may consist of a single dose or a plurality ofdoses over a period of time. Optimal dosages may be readily determinedby routine methods.

In another aspect, the invention contemplates use of an inhibitor ofIL-13 expression in the production of a medicament for treatment ofIL-13 dependent Hodgkin and Reed Sternberg cell proliferation. Asanother aspect, use of an inhibitor of IL-13 receptor biologicalactivity in the production of a medicament for treatment of IL-13dependent Hodgkin and Reed Sternberg cell proliferation is alsoprovided. The invention also embraces use of an inhibitor of IL-13receptor expression in the production of a medicament for treatment ofIL-13 dependent Hodgkin and Reed Sternberg cell proliferation. In stillanother aspect, the invention comprehends use of an inhibitor of IL-13biological activity in the production of a medicament for treatment ofIL-13 dependent Hodgkin and Reed Sternberg cell proliferation.

While the invention is exemplified with respect to expression andbiological activity of IL-13 and its cognate receptor, the inventionalso comprehends materials and methods to modulate expression andbiological activity of other compounds (and their receptors whereapplicable) demonstrated herein to have high expression levels in H/RScells, including for example, IL-5, omithine decarboxylase (ODC),ICAM-3, urokinase (UPA), IgE Fc receptor II, insulin-like growth factor(IGF) II, NF-IL3/E4BP4, Notch2, GM-CSF, interferon regulatory factors(IRF) 1, 5 and 6, nitric oxide synthase (NOS) 3, and the T cell receptordelta chain. The invention also comprehends materials and methods tomodulate expression and biological activity of compounds in IL-13pathways that participate, directly or indirectly, in H/RS cellproliferation

DETAILED DESCRIPTION OF THE INVENTION

The present invention is exemplified in the following examples. Example1 describes identification of genes differentially expressed in H/RScells. Example 2 describes Northern analysis and ELISA to confirmresults observed in Example 1. Example 3 provides even more confirmatoryevidence of differential gene expression using in situ hybridization.Example 4 demonstrates that neutralization of IL-13 activity modulatesH/RS cell proliferation.

EXAMPLE 1 Gene Expression in HD Cells

In a first series of experiments, assays were carried out to compareoverall gene expression in a HD-derived cell type compared to a normalcell line in an attempt to identify genes that are over-expressed in theHD cells. Microarray analysis has recently been developed [Schena, etal., Science 270:467-470 (1995); Schena, et al. Proc Natl Acad Sci USA93:10614-10619 (1996); Shalon, et al., Genome Res 6:639-645 (1996)] as avery efficient means of studying the differential expression of manygenes simultaneously. Gene expression in two different samples iscompared using competitive hybridization of two probes labeled withdifferent fluorescent dyes.

In the first experiments, cDNA segments from genes relevant toinflammation and neoplasia were used to examine gene expression patternsin HD-derived cell lines. Microarray expression patterns in theaneuploid, clonal cell lines L428 and KMH2 [Drexler, Leukemia andLymphoma. 9:1-25 (1993)], which were cultured from Hodgkin tissues andare regarded as HD cell lines, were compared with microarray expressionpatterns in control LCL-GK cells, an Epstein Barr virus (EBV) infectedlymphoblastoid B cell line from a healthy individual. Additional controlcells included EBV negative, non-Hodgkin lymphoma (NHL) cell lines Ly4,Ly7, and Lyl3.2. The cell lines were grown in IMDM medium supplementedwith penicillin, streptomycin, and 10% fetal calf serum.

From each cell type, poly(A)⁺ mRNA was prepared from total RNA usingOligotex-dT resin (QIAGEN, Chatsworth, Ga.) according to themanufacturer's suggested protocol. Each mRNA sample was converted intofluorescein-labeled cDNA probes using the microarray (GEM) probelabeling kit (Synteni, Palo Alto, Calif., USA), also according tomanufacturer's suggested protocol. The microarray used in theseexperiments contained 950 human genes involved in inflammation andneoplasia (Synteni, Palo Alto, Calif., USA). Two microarrays wereindependently probed with 200 ng of a 1:1 mixture of fluorescein-labeledcDNA from HD-derived L428 cells and control LCL-GK cells on microarray 1(M1) or from HD-derived KMH2 cells and LCL-GK cells on microarray 2(M2). Known concentrations of mRNA synthesized from inter-ORF regionsfrom S. cerevisiae were used as quantitation standards. Thehybridization was performed as previously described [Schena, et al.,1995. Science 270:467-470 (1995)].

Of the 950 genes displayed on the microchip, those showing greater thanthree-fold difference in expression in both HD-derived cell lines were:IL-13, IL-5, ornithine decarboxylase (ODC), ICAM-3, urokinase (UPA), IgEFc receptor II, insulin-like growth factor (IGF) II, NF-IL3/E4BP4,Notch2, GM-CSF, interferon regulatory factors (IRF) 1, 5 and 6, nitricoxide synthase (NOS) 3, and the T cell receptor delta chain. IL-13expression in HD-derived L428 and KMH2 cells was increased over that incontrol LCL-GK cells 26.7- and 17.1-fold, respectively, while IL-5expression was increased by 14.2- and 18.5-fold, respectively. Inaddition, mRNA for the IL-13 receptor was found to be expressed in bothHD-derived cell lines tested.

These results clearly demonstrate that HD-derived cell lines and H/RStumor cells express elevated levels of IL-13. Because IL-13 is known topromote IgE class switching, the elevation of IgE in H/RS cells and inthe serum of HD patients could be explained if IL-13 secreted by H/RScells affects both the H/RS cells themselves and bystander B cells.

As discussed above, the microarray hybridization showed that theexpression of NF-L3 and Notch2 was upregulated by more than 3-fold inNHL-derived cell lines. The basic leucine zipper (b-ZIP) transcriptionfactor NF-L3 acts downstream of IL-13 and has been shown to preventapoptosis after forced expression in an IL-13-dependent pro-B cell line[Ikushima, et al., Proc Natl Acad Sci USA 94:2609-2614 (1997)]. Therelevance of NF-L3 in HD is unknown.

Notch 2 is a transmembrane receptor that has been shown to be involvedin cell fate decisions [Robey, Curr Opin Genet Dev 7:551-7. (1997)]. Thehuman Notch2 gene has been mapped to chromosome 1 at position 1p13-p11,which is a region of translocations associated with neoplasia [Larsson,et al., Genomics 24:253-58 (1994)]. Chromosome 1 has also been linked tothe HD phenotype, since structural rearrangements of chromosome 1 arefrequently observed in HD [Thangavelu, et al., Hematol Oncol Clin NorthAm 3:221-36 (1984)].

Interestingly, the gene for CD30, which was first identified in theHD-derived cell line L428 [Schwab, et al., Nature 299:65-67 (1982)] andis considered a marker for the disease, is also located on chromosome 1at 1p36 [Fonatsch, et al., Genomics 14:825-6 (1992)]. CD30 is highlyexpressed in lymphoblastoid and H/RS cells and has been shown to promotea Th2 phenotype [Del Prete, et al., J Exp Med 182:1655-61 (1995)]. Thepossible roles of NF-IL3 and Notch2, and their relationship to IL-13 andthe CD30 antigen in the pathogenesis of HD lymphoma merit furtherinvestigation.

EXAMPLE 2 Northern Analysis and Cytokine Production

To evaluate the significance of the results from Example 1, Northernblots and ELISA were used to examine expression of IL-13, IL-5, GM-CSF,NF-IL3, Notch2, and urokinase in the HD-derived EBV negative cell linesL428, KMH2, and HDLM2 [Drexler, Leukemia and Lymphoma. 9:1-25 (1993)].Control cell types included lymphoblastoid EBV-infected B cell linesLCL-GK and LCL-HO, and the three EBV-negative non-Hodgkin lymphoma(NHL)-derived cell lines with either B cell (Ly4, Ly7) or T cell(Lyl3.2) phenotype.

In the Northern blot analysis, total RNA was isolated using Trizolreagent (Gibco BRL, Gaithersburg, Md.) according to the manufacturer'ssuggested protocol and 15 μg of total RNA was separated on a 1%formaldehyde agarose gel. Samples were run in 0.02 M MOPS(3-[N-morpholino]propanesulfonic acid), pH 7.0, 8 mM sodium acetate, and1 mM EDTA. The RNA was blotted overnight in 10×SSC onto a Hybond N+nylon membrane (Amersham Pharmacia Biotec, Arlington Heights, Ill., USA)and crosslinked to the membrane using UV irradiation using standardtechniques. Hybridization was carried out at 65° C. using α-[³²P]dATP-labeled oligonucleotides specific for the human Notch2, NF-IL3,urokinase, IL-13, and human β-actin genes. Probe labeling was carriedout using a Multiprime DNA labeling system (Amersham Pharmacia Biotec,Arlington Heights, Ill., USA) according to the manufacturer's suggestedprotocol. Membranes were washed once at 65° C. for 30 minutes in 2×SSCcontaining 0.1% SDS, and then at 65° C. for 30 minutes in 0.2×SSCcontaining 0.1% SDS.

In the ELISA, cell culture supernatant (10⁵ cells equivalent/ml) fromeach cell type was recovered 48 hours after medium exchange and assayedfor IL-5, IL-13, and GM-CSF production using Quantikine kits (R&DSystems, Minneapolis, Minn., USA) specific for each cytokine, accordingto the manufacturer's suggested protocol.

Results indicated that Notch2, urokinase, and NF-IL3 were up-regulatedin LCL or NHL cell lines, as well as in the HD-derived cell lines, butoverexpression of IL-13, IL-5 and GM-CSF was restricted to theHD-derived cell lines. IL-5 and GM-CSF expression and secretion could bedemonstrated only in L428 and KMH2 cells, but mRNA expression andsecretion of IL-13 could be detected in all three HD-derived cell lines.HDLM2 cells secreted a moderate amount of IL-13 (27 pg/ml), while LA28and KMH2 cells secreted significantly higher levels of (4800 pg/ml and6100 pg/ml, respectively).

EXAMPLE 3 In Situ Hybridization

To confirm that IL-13 was expressed by the rare H/RS tumor cells, insitu hybridization was carried out with sense and antisense RNA probesfor IL-13 using lymph node biopsies from four untreated patients, newlydiagnosed with classical nodular sclerosis HD. A benign reactive lymphnode biopsy was used as a control.

For in situ hybridization, paraffin sections were mounted and fixedaccording to standard protocols. A human cDNA probe specific for IL-13was generated by PCR using RT-PCR and IL-13-specific primers set out inSEQ ID NOs: 1 and 2. PCR was carried out using standard conditions wellknown and routinely practiced in the art.

5′ GTTGACCACGGTCATTGCTCTCACT SEQ ID NO: 1 3′ TTCAGTTGAACCGTCCCTCGCGAASEQ ID NO: 2The 388 bp amplification product was cloned into the pCRII vector (TAcloning kit, Invitrogen, Carlsbad, Calif., USA). Sense and antisenseprobes were synthesized from the linearized vector with SP6 or T7polymerase, labeled with ³²P-UTP, and processed as previously described[Hui, et al., Nat Genet 3:241-246 (1993)]. The sections werecounterstained with toluidine blue using a standard protocol.

The results confirmed strong and specific IL-13 expression bymorphologically identifiable H/RS cells in all patients, in contrast tobackground levels of IL-13 expression in non-H/RS cells and cells fromthe control biopsy.

EXAMPLE 4 Analysis of Cell Proliferation after Neutralization of IL-13

To investigate the effects of IL-13 and IL-5 on the proliferation ofH/RS cells, L428, KMH2 and HDLM2 cells were incubated with either (i)medium alone, (ii) medium containing neutralizing antibodies to IL-13 orIL-5, or (ii) isotype matched control antibodies. Proliferation wasmeasured by determining [³H]-thymidine incorporation at 24, 48, or 72hours after treatment.

HD-derived L428, KMH2, and HDLM2 cells and LCL control cells(2×10⁴/well) were cultured in 96-well flat-bottom plates for 24 hours,48 hours, or 72 hours in the presence of anti-IL-13, anti-IL-5, orisotype control antibodies at 5, 10, 20, 30, 50, 100 or 150 μg/ml. Cellswere treated either with IL-13 (0.5, 1, 5, 10, 50, 100 and 200 ng/ml),or with these same doses of IL-13 combined with 20 μg/ml anti-IL-13monoclonal antibody.

Tritium-labeled thymidine (1 μCi/well) was added to each well andincubation continued for eight hours. Cells were harvested on filtersand incorporation of [³H]-thymidine into cellular DNA was measured aspreviously described [Shahinian, et al., Science 261:609-12 (1993)]. Theviability of cultured cell lines after treatment with anti-IL-13neutralizing antibodies was examined by 7-aminoactinomycin D (7-AAD)staining.

Results showed no significant differences in viability betweenHD-derived cells and controls. Neutralizing antibodies against IL-13 andIL-5 had no effect on the proliferation of control LCL-HO cells. After72 hours treatment with 20 μg/ml anti-IL-13 neutralizing antibody,however, proliferation of HDLM2 cells (which secrete moderate levels ofIL-13) was suppressed to 27% of that of untreated control cells.Treatment of L428 and KMH2 cells with up to 150 μg/ml anti-IL13 antibodyhad no effect on the proliferation of the cell lines, perhaps because oftheir vigorous secretion of IL-13. No significant changes inproliferation were observed in control groups or HDLM2 cells treatedwith either isotype control antibodies or an anti-IL-5 neutralizingantibody. A combination of anti-IL-13 and anti-IL-5 antibodies did notinhibit proliferation to any greater extent than anti-IL-13 alone.Treatment with increasing concentrations of anti-IL-13 antibodydemonstrated that the effect on the proliferation of HD-derived cellswas dose-dependent. Furthermore, the anti-proliferative effect ofanti-IL-13 on HDLM2 cells could be overcome by the addition of exogenousIL-13. Treatment of HDLM2 cells with exogenous IL-13 alone did notresult in an increase in proliferation over that of untreated cells,suggesting that the cells produce saturating levels of IL-13 sufficientto support maximal proliferation.

Numerous modifications and variations in the invention as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the invention. References cited herein areincorporated by reference in their entireties.

1.-6. (canceled)
 7. A method for inhibiting IL-13 dependentproliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprisingthe step of contacting the H/RS cells with a compound that inhibitsexpression of IL-13.
 8. The method of claim 7 wherein the compound thatinhibits IL-13 expression is an antisense polynucleotide thatspecifically recognizes a polynucleotide encoding IL-13.
 9. The methodof claim 7 wherein the compound that inhibits IL-13 expression is aribozyme that specifically recognizes and acts on a polynucleotideencoding IL-13.
 10. A method for inhibiting IL-13 dependentproliferation of Hodgkin and Reed Sternberg (H/RS) cell types comprisingthe step of contacting the H/RS cells with a compound that inhibitsexpression of IL-13 receptor.
 11. The method of claim 10 wherein thecompound that inhibits IL-13 receptor expression is an antisensepolynucleotide that specifically recognizes a polynucleotide encodingthe IL-13 receptor.
 12. The method of claim 10 wherein the compound thatinhibits IL-13 expression is a ribozyme that specifically recognizes andacts on a polynucleotide encoding IL-13. 13-20. (canceled)
 21. A methodto identify a compound that inhibits IL-13 dependent proliferation ofHodgkin and Reed Sternberg (H/RS) cell types comprising the steps of: a)growing H/RS cell types b) measuring IL-13 expression c) incubating thecells with a candidate inhibitor compound d) measuring IL-13 expressionwith the candidate compound e) identifying the candidate compound as aninhibitor when lower levels of IL-13 expression are measured with thecandidate compound than in an absence of the candidate compound.
 22. Themethod according to claim 21 wherein the candidate inhibitor compound isan anti-sense polynucleotide that specifically binds to a polynucleotideencoding IL-13.
 23. The method of claim 21 wherein the candidateinhibitor compound is a ribozyme that specifically recognizes and actson a polynucleotide encoding IL-13. 24-26. (canceled)
 27. A method fortreating Hodgkin's Disease comprising the steps of administering to anindividual in need thereof a therapeutically effective amount of acompound that inhibits IL-13 expression.
 28. The method according toclaim 27 wherein the inhibitor is an antisense polynucleotide thatspecifically binds a polynucleotide encoding IL-13.
 29. The method ofclaim 27 herein the candidate inhibitor compound is a ribozyme thatspecifically recognizes and acts on a polynucleotide encoding IL-13.30-32. (canceled)
 33. A method for treating Hodgkin's Disease comprisingthe step of administering to an individual in need thereof atherapeutically effective amount of a compound that inhibits IL-13receptor expression. 34-39. (canceled)